Mechanism of bleomycin: evidence for 4'-ketone formation in poly(dA-dU) associated exclusively with free base release

Incubation of poly(dA-[3'-3H]dU), poly(dA-[5'-3H]dU), or poly(dA-[5'-3H]dT) under a variety of conditions with activated bleomycin resulted in the production of free nucleic acid base, base propenal, and a small amount of 3H2O. Adjustment of the terminated reaction mixture to pH 10 and incubation at 95 degrees C resulted in a time-dependent increase in 3H2O to an amount equal to the amount of free base. If the terminated reaction mixture was incubated with NaBH4 prior to the heat and alkaline treatment, the release of 3H2O was significantly inhibited. These results are consistent with the generation by activated bleomycin of a 4'-ketone yielding free base, with the exchange of the 3'- and 5'-hydrogens by enolization and with the alkaline-induced strand scission occurring from this intermediate.